Using new Next Generation Sequencing (NGS) approaches to analyze the fidelity of HIV reverse transcription in Endogenous Reverse Transcription reactions (ERT)

使用新的下一代测序 (NGS) 方法来分析内源性逆转录反应 (ERT) 中 HIV 逆转录的保真度

• 批准号: 10759845
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 22.51万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-20 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10759845
• 关键词: APOCEC3G gene; Address; Affect; Area; Biological Assay; Capsid; Cell Culture Techniques; Cell Nucleus; Cells; Cellular Assay; Complement; Consensus; Coupled; DNA; DNA biosynthesis; DNA-Directed RNA Polymerase; Data; Defense Mechanisms; Development; Divalent Cations; Environment; Enzymes; Evolution; Future; Genetic Complementation Test; Genetic Variation; Genome; Goals; HIV; HIV-1; Health; Human; Immune response; Immunity; In Vitro; Kinetics; Lead; Letters; Modernization; Mutagenesis; Mutation; Mutation Analysis; Nucleotides; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Polymerase; Positioning Attribute; Process; Production; Proteins; Publications; RNA; RNA Polymerase II; RNA-Directed DNA Polymerase; Reaction; Reporting; Research Personnel; Retroviridae; Reverse Transcription; Ribavirin; Role; Sodium Chloride; Source; System; Time; Variant; Viral; Virion; Virus; Virus Replication; Work; ds-DNA; experimental study; improved; in vitro Assay; in vitro activity; in vivo; next generation sequencing; novel; tool

Analysis of RTs from other retroviruses in vitro suggests that HIV RT has exceptionally low fidelity. For example, MuLV-RT shows ~5-fold greater fidelity than HIV RT on both RNA and DNA templates across several publications from different groups, and even in identical experiments in the same publication. Yet cellular replication data indicates that these viruses have similar mutation rates. The mutation profile observed in cellular assay with HIV is also inconsistent with in vitro results. Recently, our lab and others have found that the in vitro fidelity of HIV RT improves when physiological concentrations of free Mg2+ (~0.25-1 mM) are used as opposed to the higher (5-10 mM) concentration optimized for in vitro activity that have typically been used in fidelity assays in vitro . This may be one factor, possibly among many, that explains differences between HIV RT and other viral RTs. MuLV and AMV RTs, for example, do not show significantly improved fidelity in lower Mg2+ and MuLV RT has an error rate in vitro that is more consistent with cellular replication values. The lack of correlation with respect to the overall mutation rate and spectrum of mutations observed in vitro vs. cellular replication has made it impossible to determine RT’s contribution to the mutation spectrum observed in cells. This in turn makes it difficult to assess the contributions of mutagenic cellular factors (e.g., APOBEC3G, UNG, and RNA polymerase II) to the observed spectrum. A thorough understanding of the factors that contribute to the genetic diversity of HIV will require a better understanding of HIV RT’s role in the process, and a clear definition of the mutation rate and spectrum observed with HIV RT in vitro, and during virus replication in cells. Understanding how HIV generates genetic diversity, which fuels the production of new viruses that can escape immunity and drug therapy, has been a long-standing goal of HIV researchers. We believe this proposal will move us forward toward this goal. New high fidelity Next Generation Sequencing (NGS) approaches, including Single Strand Consensus Sequencing (SSCS) and duplex sequencing, allow the rapid analysis of thousands of mutations in a single experiment and has brought in a new era for mutational analysis. The same can be said for recent findings from Dr. Wes Sundquist’s lab, our collaborator on this work, that demonstrate the complete replication of HIV dsDNA in a highly efficient cell-free virion-based endogenous reverse transcription (referred to as “ERT” in this proposal) system. By applying modern NGS approaches to DNA synthesis with purified RT in vitro, in ERT reactions, and during virus replication in cells, the mutation rates and spectrums of these processes can be directly compared. Notably, this will be the first fidelity analysis in the novel ERT system. This will more clearly define RT’s role and relative contribution to HIV mutagenesis, and the roles of virion and cellular factors. Specific Aims: Aim 1: To use Next Generation Sequencing (NGS) to compare the mutation rates and spectrums from purified HIV RT and highly efficient Endogenous Reverse Transcription (ERT) assays; Aim 2: To compare the rate and spectrum of mutations made by purified RT and in ERT reactions, to those observed during reverse transcription in cells.

对其他逆转录病毒的 RT 进行体外分析表明，HIV RT 的保真度极低。 MuLV-RT 在多个 RNA 和 DNA 模板上的保真度比 HIV RT 高约 5 倍 来自不同组的出版物，甚至同一出版物中的相同实验。 复制数据表明这些病毒具有在细胞中观察到的相似突变率。 最近，我们实验室和其他人发现，HIV的体外检测结果也不一致。 当使用生理浓度的游离 Mg2+ (~0.25-1 mM) 时，HIV RT 的保真度会提高 至针对体外活性进行优化的较高浓度 (5-10 mM)，通常用于保真度测定 这可能是解释 HIV RT 与其他病毒之间差异的众多因素之一。 例如，MuLV 和 AMV RT 在较低 Mg2+ 和 MuLV RT 中没有表现出显着改善的保真度。 体外错误率与细胞复制值缺乏相关性。 关于体外观察到的总体突变率和突变谱与细胞复制的比较， 无法确定 RT 对细胞中观察到的突变谱的贡献。 难以评估致突变细胞因素（例如 APOBEC3G、UNG 和 RNA 聚合酶）的贡献 II）对观察到的谱的全面了解，导致遗传多样性的因素。 HIV需要更好地了解HIV RT在此过程中的作用，并明确突变率的定义 以及在体外和细胞内病毒复制过程中观察到的病毒谱。 产生遗传多样性，从而促进可以逃避免疫和药物的新病毒的产生 治疗，一直是艾滋病毒研究人员的长期目标，我们相信这一提议将推动我们朝着这个方向前进。 新的高保真下一代测序 (NGS) 方法，包括单链共识。 测序 (SSCS) 和双工测序可快速分析单个突变中的数千个突变 实验并为突变分析带来了一个新时代，最近的发现也是如此。 Wes Sundquist 博士的实验室是我们这项工作的合作者，该实验室展示了 HIV dsDNA 的完整复制 高效的基于无细胞病毒粒子的内源逆转录（在本提案中称为“ERT”） 通过应用现代 NGS 方法在体外、ERT 反应和纯化 RT 中进行 DNA 合成。 病毒在细胞内复制期间，可以直接比较这些过程的突变率和谱。 值得注意的是，这将是新型 ERT 系统中的首次保真度分析，这将更清楚地定义 RT 的作用和内容。 对 HIV 突变的相对贡献，以及病毒粒子和细胞因子的作用。 具体目标： 目标 1：使用。 下一代测序 (NGS) 用于比较纯化的 HIV RT 和 HIV 的突变率和谱 高效内源逆转录 (ERT) 检测；目标 2：比较 纯化 RT 和 ERT 反应中产生的突变，与细胞逆转录过程中观察到的突变。